Many natural products with antibiotic, anticancer and antifungal properties are synthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), yet most microbes do not express the great majority of their NRPS or PKS enzymes. In the first granting period of this R01, we developed mass spectrometry (MS) into a facile approach to directly detect covalent intermediates in ~30 systems that have been studied in vitro. We propose to continue this activity for peptide antibiotics and iterative PKS systems (Specific Aim 1). In Specific Aims 2 and 3, we also propose to streamline NRPS/PKS assays using high throughput LC-MS. This blends aspects of enzymology with microbial proteomics of native producers into an entirely new approach to natural product discovery and the study of NRPS and PKS systems. By virtue of their huge size (often >2000 amino acids) and unique marker ions deriving from their common cofactor (phosphopantetheine), we show proof-of-concept for mass spectrometry-based proteomics with highly selective detection of expressed NRPS/PKS gene clusters in native proteomes - without requiring genome sequence information a priori. This method (dubbed PrISM) will allow unambiguous detection of NRPS/PKS systems in Bacilli, Streptomyces, and even fungi. We therefore advocate a new proteome-first strategy to find gene clusters that are actually expressed and efficiently link them to the natural products they produce. The new approaches proposed here are especially timely in an era when new types of mega-enzyme assembly lines that defy classification and cloning are being discovered at an accelerating rate, but relatively few new approaches are available to study them. With emphasis on this area of natural products expanding, our activities will assist in the fundamental understanding of chemical processes involved in biogenesis of new natural products of the types that have provided so many of our societies useful drugs to fight both bacterial infection and cancer. PUBLIC HEALTH RELEVANCE: The discovery of new and novel natural products is burdened by the rediscovery of common antibiotics and antiproliferative compounds. Compounds made by NRPS/PKS enzymes have proven to provide diverse modes of action toward myriad different targets. The use of proteomics technology to discover natural products is a novel approach to this difficult problem.